In analytical chemistry, in particular in clinical chemical diagnostics, there is a continuously increasing demand for enzymatic methods for the determination of natural products, biological metabolic products and compounds derived therefrom. The reasons are the extraordinarily high specificity of enzyme-catalyzed substance conversions, their rapid and defined course which is free from losses under mild reaction conditions--usually between 15.degree. and 40.degree. C. in aqueous media in a neutral pH range--as well as the possibility of determining these quantitatively in a simple and sensitive manner, in particular using photometric measurement procedures either directly or with the aid of coupled indicator reactions.
An enzymatic method of determination is also particularly valuable for the determination of creatinine, a component of serum and urine that is important for kidney diagnosis, which can be converted in an enzymatic reaction which has been known for a long time into 1-methylhydantoin (N-methylhydantoin, NMH) and ammonia by means of creatinine iminohydrolase (E.C. 3.5.4.21).
Several methods are already known for the enzymatic determination of creatinine in serum or urine (Wahlefeld, A. W.; G. Holz and H. U. Bergmeyer, in H. U. Bergmeyer: Methoden der enzymatischen Analyse, Third Edition, Volume II, Verlag Chemie, Weinheim 1974, p. 1834-1838; Fossati, P.; L. Prencipe, and G. Berti, Clinical Chemistry 29, 1494-1496 (1983); Tanganelli, E.; L. Prencipe; D. Bassi; S. Cambiaghi, and E. Murador, Clinical Chemistry 28, 1461-1464 (1983); however, they all have the disadvantage that they either proceed via creatine (Wahlefeld et al.; Fossati et al.) or via ammonia (Tanganelli et al.) as intermediate products in the reaction sequence. These are substances which are present in the serum or urine sample to be analyzed in varying concentrations which are quite substantial compared to creatinine. Thus in order to determine creatinine in these cases, it is necessary to carry out differential measurements on two separate or successive reaction mixtures, one in which first the free creatine or ammonia is determined and a second in which the portion of additional creatine or ammonia formed from creatinine is determined ("sample/sample blank method" or "A.sub.1 /A.sub.2 method") by addition of either creatinine amidohydrolase (E.C. 3.5.2.10) or creatinine iminohydrolase (=creatinine deiminase).
Such methods are relatively complicated for a manual procedure and their application to automated analytical systems is also very limited, in particular when longer incubation periods are necessary for the completion of the conversion reactions. In principle it is possible to carry out the creatinine determination with the so-called kinetic "fixed-time" method by suitable choice of reaction conditions using the known enzymatic methods and thus obviating the sample blank measurement; this, however, requires very strict adhesion to the measurement times under defined temperature conditions which is only possible with an adequate precision on automated analyzers and conversely virtually excludes manual application.
A new enzymatic method for the determination of creatinine or 1-methylhydantoin is described in U.S. Pat. No. 4,816,393. The substance 1-methylhydantoin is hydrolyzed to N-carbamoylsarcosine using the enzyme 1-methylhydantoinase (NMHase) which requires a nucleoside triphosphate, preferably ATP, as well as divalent metal ions and in some circumstances K.sup.+ ions and/or NH.sub.4 ions for its activity. This enzyme is also described in H. Yamada et al., FEBS Microbiol. Letters 30, 337-340 (1985), in S. Shimizu et al., Arch. Microbiol. 145, 322-328 (1986) and in Shimizu et al., Biochem. Biophys. Res. Comm. 142, 1006-1012 (1987).
It is available for example from Arthrobact. spec., Micrococcus spec., Moraxella spec., or Brevibacterium spec.
In a preferred variant of the test N-carbamoylsarcosine amidohydrolase (E.C. 3.5.3.59) known from U.S. Pat. No. 4,645,739 converts N-carbamoylsarcosine to sarcosine which can then, together with e.g. sarcosine oxidase (E.C. 1.5.3.1) and peroxidase (E.C. 1.11.1.7), be detected photometrically, e.g. by a suitable redox indicator: creatinine+H.sub.2 O.fwdarw.1-methylhydantoin+NH.sub.3 1-methylhydantoin+ATP+2H.sub.2 O.fwdarw.N-carbamoylsarcosine+ADP+P.sub.i N-carbamoylsarcosine+H.sub.2 O.fwdarw.sarcosine+NH.sub.3 +CO.sub.2 sarcosine+O.sub.2 +H.sub.2 O.fwdarw.H.sub.2 O.sub.2 +formaldehyde+glycine H.sub.2 O.sub.2 +redox indicator (red.).fwdarw.H.sub.2 O+redox indicator (ox.)
A corresponding photometric method for the determination of creatinine is also described in J. Siedel et al., Anal. Letters 21, 1009-1017 (1988).
Endogenous substances present in body fluids should not interfere with this creatinine or 1-methylhydantoin determination since 1-methylhydantoin and the reaction products of the subsequent indicator reaction are not natural components of serum or urine. Therefore a sample blank measurement should not be necessary.
However, it has turned out that the use of NMHase for creatinine determination is limited by two disadvantageous enzyme properties. NMHase requires enzyme-bound 1-methylhydantoin for its stability (ca. 2 -2.5 .mu.mol/KU, U=international unit), i.e. it is stabilized by its own substrate. In general 1-methylhydantoin remains bound-to the enzyme during enzyme purification. However, in the presence of a nucleoside triphosphate and divalent metal ions, such as Mg.sup.2+, as for example in a method for determining creatinine it is completely degraded enzymatically and causes a blank reaction similar to an endogenous substrate.
This blank reaction is especially disadvantageous in a determination method in which the amount of NMHase used in the test can vary widely, which can be the case for example in a carrier-bound test. Since the blank reaction caused by enzyme-bound NMH depends, on the one hand on the amount of NMHase used and on the other hand also on the varying NMH content of the NMHase itself, it is also necessary to always determine the blank reaction separately for such a test and this blank value has to be subtracted from the measured value.
On the other hand the NMHase is very unstable without enzyme-bound substrate and cannot be used for enzymatic methods of determination such as e.g. creatinine determination. In addition it was found that the nucleoside triphosphate necessary for the enzymatic reaction of NMHase is only of limited stability in the presence of NMHase since an NMHase shows ATPase activity and degrades ATP over the course of time.
The object of the present invention was therefore to provide a process which stabilizes NMHase in such a way that a stable enzyme is provided even in the absence of enzyme-bound 1-methylhydantoin or other substrate analogues and to provide an improved and more exact method for the determination of creatinine or other analytes which can be detected by means of a reaction catalyzed by NMHase.
This object is achieved according to the present invention by the measures as described in the claims.